The availability of free arachidonate is critical to the production of prostaglandins in platelets, and is, therefore essential to platelet function. Phospholipase A2 (PLA2) the enzyme primarily responsible for regulation of arachidonate is known to be activated, but the mechanism(s) of activation and role of this enzyme is poorly understood. This proposal examines the role of PLA2 in human platelet function. We will solubilize and purify this enzyme by sulfuric acid extraction, pH precipitation, and affinity chromatography. PLA2 activity toward synthetic phospholipid substrates containing 14C-fatty acids will be determined. The enzyme-will be characterized with respect to intracellular localization, isoelectric point, amino acid composition, phospholipid-fatty acid substrate specificity, ion requirements (Ca 2 ion, Mg2 ion, Mg2 ion), inhibitors (i.e. antiinflammatory agents, intracellular proteins) and stimulators (i.e., cations, acidic phospholipids). The physical and chemical parameters (i.e., zeta potential, surface pressure and phase transition) or phospholipid substrates that may influence the susceptibility to hydrolysis of phospholipids in biological membranes will also be studied. We will examine the effect of in vitro moderators on endogenous PLA2 activity in platelet subcellular fractions and intact cells. Endogenous phospholipid hydrolysis will be monitored by quantitative thin layer and gas liquid chromatography. We hope to correlate PLA2-induced alterations of specific phospholipids with alterations in platelet function as measured by platelet aggregation and release, clotting time, and clot retraction. Similarly, we will examine the effect of exogenously added PLA2 on platelet function. Our goal is to understand the mechanism(s) by which the expression of platelet PLA2 activity is modulated in biological membranes. With a knowledge of physiologic factors and pharmacologic agents that regulate activation of PLA2 in platelet membranes, similar responses (i.e. inflammation) that occur in other tissues or cells may be controlled.